Using subtractive hybridization, we have isolated a novel neuropeptide gene which is expressed exclusively in a subregion of the posterior hypothalamus. Hypocretin contains a eukaryotic secretory signal sequence and encodes two neuropeptides, 38 and 28 amino acids in length, which are conserved between mouse, rat, and human, indicating an evolutionarily conserved function for these novel neuropeptides. We have raised a high quality antiserum to the hypocretin neuropeptide precursor, localized cell bodies within the hypothalamus, and begun mapping of the terminal projections of hypocretin-containing neurons. We have also obtained evidence that these novel peptides affect neuronal excitability and intracellular calcium levels in vitro and body temperature in vivo. Other investigators have isolated these same molecules which they called the orexins and demonstrated that the affect food intake. Based on these results, we hypothesize that the hypocretin peptides play a role in regulating energy balance and metabolism. We propose to characterize further this novel neurotransmitter pathway by describing the phenotype of neurons which express hypocretin in the hypothalamus and identifying the neuroanatomical regions to which hypocretin-containing neurons project. In in vitro studies, we will characterize the response of neurons to hypocretin peptidergic stimulation and determine the cellular electrophysiological response of neurons to hypocretin application. We will also conduct in vivo studies in which we will obtain functional information about the effects of hypocretin neuropeptides on brain activity and test the hypothesis that hypocretin peptides play a CNS role in regulating energy balance and metabolism. This proposal presents an exciting, integrated research program to characterize a heretofore unstudied neurotransmitter system expressing this novel set of neuropeptides and to determine the effects of these peptides in vitro and in vivo.